Serine proteases are involved in numerous biological processes, including apoptosis, blood coagulation, viral maturation, and cancer. Highly specific protease inhibitors can be powerful tools for studying proteases and elucidating their in vivo roles. Ecotin, a macromolecular serine protease inhibitor found in the periplasm of E. coli, offers a unique platform for protein engineering. In contrast to most protease inhibitors, ecotin shows very broad specificity and also binds as a dimer to target proteases, forming a tetrameric complex involving ecotin-protease recognition both at the active site and at a distal secondary site. Alanine shaving and phage display experiments on several proteases have indicated that the importance of each of ecotin's two binding sites differs with the specific protease and have also shown that ecotin can be remodeled to make a more potent inhibitor. Using current methods of protein engineering, we intend to create variants of ecotin that will be potent and specific inhibitors of serine proteases, such as alpha lytic protease, hepatitis A virus 3C protease, yellow fever NS3 protease, or proteases involved in cancer. Utilizing known crystal structures, we will use computer graphics to model the interaction between ecotin and target proteases. Structure-guided loop-trimming and specificity tuning will be used to convert ecotin into a potent inhibitor of these serine proteases. Crystal structures of these ecotin variants complexed with their target proteases will be analyzed to understand the mechanism of protease inhibition. By combining a structure-based approach with the power of combinatorial libraries, we expect to evolve protease inhibitors that will be able to elucidate the roles of proteases in vivo, help understand the nature of protease inhibition and molecular interaction, and perhaps lead to new therpies for life-threatening diseases.